Multivalent Meningococcal Polysaccharide-Protein Conjugate Vaccine

ABSTRACT

The present invention describes a combined vaccine that offers broad protection against meningococcal disease caused by the pathogenic bacteria  Neisseria meningitidis.  The vaccine is comprised of four distinct polysaccharide-protein conjugates that are formulated as a single dose of vaccine. Purified capsular polysaccharides from  Neisseria meningitidis  serogroups A, C, W-135, and Y are chemically activated and selectively attached to a carrier protein by means of a covalent chemical bond, forming polysaccharide-protein conjugates capable of eliciting long-lasting immunity to a variety of  N. meningitidis  strains in children as well as adults.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/855,994, filed Sep. 16, 2015, which is a continuation of U.S.application Ser. No. 14/636,870, filed Mar. 3, 2015, now U.S. Pat. No.9,173,955; which is a continuation of U.S. application Ser. No.14/257,551, filed Apr. 21, 2014, now U.S. Pat. No. 8,999,354, which is acontinuation of U.S. application Ser. No. 13/738,698, filed Jan. 10,2013, now U.S. Pat. No. 8,741,314, which is a divisional of U.S.application Ser. No. 10/054,638, filed Jan. 22, 2002, now U.S. Pat. No.8,722,062, which claims the benefit of U.S. Provisional Application No.60/263,435, filed Jan. 23, 2001, the entire contents of all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the invention

The present invention relates to the field of medicine generally, andmore specifically to microbiology, immunology, vaccines and theprevention of infection by a bacterial pathogen by immunization.

Summary of the Related Art

Neisseria meningitidis is a leading cause of bacterial meningitis andsepsis throughout the world. The incidence of endemic meningococcaldisease during the last thirty years ranges from 1 to 5 per 100,000 inthe developed world, and from 10 to 25 per 100,000 in developingcountries (Reido, F. X., et. al. 1995). During epidemics the incidenceof meningococcal disease approaches 1000 per 100,000. There areapproximately 2,600 cases of bacterial meningitis per year in the UnitedStates, and on average 330,000 cases in developing countries. The casefatality rate ranges between 10 and 20%.

Pathogenic meningococci are enveloped by a polysaccharide capsule thatis attached to the outer membrane surface of the organism. Thirteendifferent serogroups of meningococci have been identified on the basisof the immunological specificity of the capsular polysaccharide (Frasch,C. E., et. al. 1985). Of these thirteen serogroups, five cause themajority of meningococcal disease; these include serogroups A, B, C,W135, arid Y. Serogroup A is responsible for most epidemic disease.Serogroups B, C, and Y cause the majority of endemic disease andlocalized outbreaks.

The human naso-oropharyngeal mucosa is the only known natural reservoirof Neisseria meningitidis. Colonization takes place both at the exteriorsurface of the mucosal cell and the subepithelial tissue of thenasopharynx. Carriage of meningococci can last for months. Spreading ofmeningococci occurs by direct contact or via air droplets. Meningococcibecome invasive by passing through the mucosal epithelium via phagocyticvacuoles as a result of endocytosis. Host defense of invasivemeningococci is dependent upon complement-mediated bacteriolysis. Theserum antibodies that are responsible for complement-mediatedbacteriolysis are directed in large part against the outer capsularpolysaccharide.

Vaccines based on meningococcal polysaccharide have been described whichelicit an immune response against the capsular polysaccharide. Theseantibodies are capable of complement-mediated bacteriolysis of theserogroup specific meningococci. The meningococcal polysaccharidevaccines were shown to be efficacious in children and adults (Peltola,H., et. al. 1977 and Artenstein, M. S., et. al. 1970), but the efficacywas limited in infants and young children (Reingold, A. L., et. al.1985). Subsequent doses of the polysaccharide in younger populationselicited a weak or no booster response (Goldschneider, I., et. al. 1973and Gold, R., et. al. 1977). The duration of protection elicited by themeningococcal polysaccharide vaccines is not long lasting, and has beenestimated to be between 3 to 5 years in adults and children above fouryears of age (Brandt, B., et. al. 1975, Kayhty, H., et. al. 1980, andCeesay, S. J., et. al. 1993). For children from one to four years oldthe duration of protection is less than three years (Reingold, A. L.,et. al. 1985).

Polysaccharides are incapable of binding to the major histocompatibilitycomplex molecules, a prerequisite for antigen presentation to andstimulation of T-helper lymphocytes, i.e., they are T-cell independentantigens. Polysaccharides are able to stimulate B lymphocytes forantibody production without the help of T-helper lymphocytes. As aresult of the T-independent stimulation of the B lymphocytes, there is alack of memory induction following immunization by these antigens. Thepolysaccharide antigens are capable of eliciting very effectiveT-independent responses in adults, but these T-independent responses areweak in the immature immune system of infants and young children.

T-independent polysaccharide antigens can be converted to T-dependentantigens by covalent attachment of the polysaccharides to proteinmolecules (“carriers” or “carrier proteins”). B cells that bind thepolysaccharide component of the conjugate vaccine can be activated byhelper T cells specific for peptides that are a part of the conjugatedcarrier protein. The T-helper response to the carrier protein serves toaugment the antibody production to the polysaccharide.

The serogroup B polysaccharide has been shown to be poorly tonon-immunogenic in the human population (Wyle, F. A., et. al. 1972).Chemical attachment of this serogroup polysaccharide to proteins has notsignificantly altered the immune response in laboratory animals(Jennings, H. J., et. al. 1981). The reason for the lack of immuneresponse to this serogroup polysaccharide is thought to arise fromstructural similarities between the serogroup B polysaccharide andpolysialylated host glycoproteins, such as the neural cell adhesionmolecules.

A meningococcal conjugate vaccine based on serogroup C polysaccharidehas been described. This monovalent vaccine elicits a strong functionalantibody response to the capsular polysaccharide present on strains ofN. meningitidis corresponding to serogroup C. Such a vaccine is onlycapable of protecting against disease caused by serogroup C bacteria.

Existing vaccines based on meningococcal polysaccharide are of limiteduse in young children and do not provide long-lasting protection inadults. The only meningococcal vaccine which has been shown to becapable of eliciting long-lasting protection in all groups, includingchildren, at risk for meningococcal infection is based on apolysaccharide from a single serogroup of N. meningitidis and providesno protection against infection by other serogroups. Thus, a need existsfor a meningococcal conjugate vaccine capable of conferring broad,long-lived protection against meningococcal disease in children andadults at risk for meningococcal infection. The multivalentmeningococcal polysaccharides of the present invention solve this needby providing vaccine formulations in which immunogenic polysaccharidesfrom the major pathogenic serogroups of N. meningitidis have beenconverted to T-dependent antigens through conjugations to carrierproteins.

SUMMARY OF THE INVENTION

The present invention provides immunological compositions for treatmentof meningococcal polysaccharide-protein conjugates caused by pathogenicNeisseria meningitidis.

The present invention provides immunological compositions comprising twoor more protein-polysaccharide conjugates, wherein each of theconjugates comprises a capsular polysaccharide from N. meningitidisconjugated to a carrier protein.

The present invention provides immunological compositions comprising twoor more distinct protein-polysaccharide conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a differentserogroup of N. meningitidis conjugated to a carrier protein.

The present invention provides vaccines for meningococcalpolysaccharide-protein conjugates caused by pathogenic Neisseriameningitidis. The present invention provides multivalent meningococcalvaccines comprised of immunologically effective amounts of from two tofour distinct protein-polysaccharide conjugates, wherein each of theconjugates contains a different capsular polysaccharide conjugated to acarrier protein, and wherein each capsular polysaccharide is selectedfrom the group consisting of capsular polysaccharide from serogroups A,C, W-135 and Y.

The present invention also provides methods of manufacture of amultivalent meningococcal polysaccharide-protein composition comprisingpurifying two or more capsular polysaccharides from pathogenic Neisseriameningitidis; conjugating the purified polysaccharides to one or morecarrier proteins and combining the conjugates to make the multivalentmeningococcal polysaccharide-protein composition.

The present invention further provides a method of inducing animmunological response to capsular polysaccharide of N. meningitidiscomprising administering an immunologically effective amount of theimmunological composition of the invention to a human or animal.

The present invention provides a multivalent meningococcal vaccinecomprised of immunologically effective amounts of from two to fourdistinct protein-polysaccharide conjugates, wherein each of theconjugates contains a different capsular polysaccharide conjugated to acarrier protein, and wherein each capsular polysaccharide is selectedfrom the group consisting of capsular polysaccharide from serogroups A,C, W-135 and Y.

The present invention provides a method of protecting a human or animalsusceptible to infection from N. meningitidis comprising administeringan immunologically effective dose of the vaccine of the invention to thehuman or animal.

All patents, patent applications, and other publications recited hereinare hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises an immunological composition of two ormore distinct protein-polysaccharide conjugates, wherein each of theconjugates comprises a capsular polysaccharide conjugated to a carrierprotein. Thus, the present invention includes compositions that comprisetwo or more different capsular polysaccharides conjugated to one or morecarrier protein(s).

Capsular polysaccharides can be prepared by standard techniques known tothose of skill in the art (ref). In the present invention capsularpolysaccharides prepared from serogroups A, C, W-135 and Y of N.meningitidis are preferred.

In a preferred embodiment, these meningococcal serogroup conjugates areprepared by separate processes and formulated into a single dosageformulation. For example, capsular polysaccharides from serogroups A, C,W-135 and Y of N. meningitidis are separately purified.

In a preferred embodiment of the present invention the purifiedpolysaccharide is depolymerized and activated prior to conjugation to acarrier protein. In a preferred embodiment of the present inventioncapsular polysaccharides of serogroups A, C, W-135, and Y from N.meningitidis are partially depolymerized using mild oxidativeconditions.

The depolymerization or partial depolymerization of the polysaccharidesmay then be followed by an activation step. By “activation” is meantchemical treatment of the polysaccharide to provide chemical groupscapable of reacting with the carrier protein. A preferred activationmethod involves treatment with adipic acid dihyrazide in physiologicalsaline at pH 5.0=0.1 for approximately two hours at 15 to 30° C. Oneprocess for activation is described in U.S. Pat. No. 5,965,714.

Once activated, the capsular polysaccharides may then be conjugated toone or more carrier proteins. In a preferred embodiment of the presentinvention each capsular polysaccharide is separately conjugated to asingle carrier protein species. In a preferred embodiment the capsularpolysaccharides from serogroups A, C, W-135 and Y of N. meningitidis areeach separately conjugated to the same carrier protein species.

Carrier proteins may include inactivated bacterial toxins such asdiphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT,E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outermembrane proteins such as, outer membrane complex c (OMPC), porins,transferrin binding proteins, pneumolysis, pneumococcal surface proteinA (PspA), or pneumococcal adhesin protein (PsaA), could also be used.Other proteins, such as ovalbumin, keyhole limpit hemocyanin (KLH),bovine serum albumin (BSA) or purified protein derivative of tuberculin(PPD) may also be used as carrier proteins. Carrier proteins arepreferably proteins that are non-toxic and non-reactogenic andobtainable in sufficient amount and purity. Carrier proteins should beamenable to standard conjugation procedures. In a preferred embodimentof the present invention diphtheria toxin purified from cultures ofCorynebacteria diphtherias and chemically detoxified using formaldehydeis used as the carrier protein.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates may be purified (enriched withrespect to the amount of polysaccharide-protein conjugate) by a varietyof techniques. One goal of the purification step is to remove theunbound polysaccharide from the polysaccharide-protein conjugate. Onemethod for purification, involving ultrafiltration in the presence ofammonium sulfate, is described in U.S. Pat. No. 6,146,902.Alternatively, conjugates can be purified away from unreacted proteinand polysaccharide by any number of standard techniques including, interalia, size exclusion chromatography, density gradient centrifugation,hydrophobic interaction chromatography or ammonium sulfatefractionation. See, e.g., P. W. Anderson, et. al. (1986). J. Immunol.137: 1181-1186. See also H. J. Jennings and C. Lugowski (1981) J.Immunol. 121: 1011-1018.

After conjugation of the polysaccharide and carrier protein theimmunological compositions of the present invention are made bycombining the various polysaccharide-protein conjugates. Theimmunological compositions of the present invention comprise two or moredifferent capsular polysaccharides conjugated to one or more carrierprotein(s). A preferred embodiment of the present invention is abivalent immunological composition comprising capsular polysaccharidesfrom serogroups A and C of N. meningitidis separately conjugated todiptheria toxoid. More preferably the present invention is a tetravalentimmunological composition comprising capsular polysaccharides fromserogroups A, C, W-135 and Y of N. meningitidis separately conjugated todiptheria toxoid.

Preparation and use of carrier proteins, and a variety of potentialconjugation procedures, are well known to those skilled in the art.Conjugates of the present invention can be prepared by such skilledpersons using the teachings contained in the present invention as wellas information readily available in the general literature. Guidance canalso be obtained from any one or all of the following U.S. patents, theteachings of which are hereby incorporated in their entirety byreference: U.S. Pat. No. 4,356,170; U.S. Pat. No. 4,619,828; U.S. Pat.No. 5,153,312; U.S. Pat. No. 5,422,427 and U.S. Pat. No. 5,445,817.

The immunological compositions of the present invention are made byseparately preparing polysaccharide-protein conjugates from differentmeningococcal serogroups and then combining the conjugates. Theimmunological compositions of the present invention can be used asvaccines. Formulation of the vaccines of the present invention can beaccomplished using art recognized methods. The vaccine compositions ofthe present invention may also contain one or more adjuvants. Adjuvantsinclude, by way of example and not limitation, aluminum adjuvants,Freund's Adjuvant, BAY, DC-chol, pcpp, monophoshoryl lipid A, CpG,QS-21, cholera toxin and formyl methionyl peptide. See, e.g., VaccineDesign, the Subunit and Adjuvant Approach, 1995 (M. F. Powell and M. J.Newman, eds., Plenum Press, NY). The adjuvant is preferably an aluminumadjuvant, such as aluminum hydroxide or aluminum phosphate.

As demonstrated below, the vaccines and immunological compositionsaccording to the invention elicit a T-dependent-like immune response invarious animal models, whereas the polysaccharide vaccine elicits aT-independent-like immune response. Thus, the compositions of theinvention are also useful research tools for studying the biologicalpathways and processes involved in T-dependent-like immune responses toN. meningitidis antigens.

The amount of vaccine of the invention to be administered a human oranimal and the regime of administration can be determined in accordancewith standard techniques well known to those of ordinary skill in thepharmaceutical and veterinary arts taking into consideration suchfactors as the particular antigen, the adjuvant (if present), the age,sex, weight, species and condition of the particular animal or patient,and the route of administration. In the present invention, the amount ofpolysaccharide-protein carrier to provide an efficacious dose forvaccination against N. meningitidis can be from between about 0.02 μg toabout 5 μg per kg body weight. In a preferred composition and method ofthe present invention the dosage is between about 0.1 μg to 3 μg per kgof body weight. For example, an efficacious dosage will require lessantibody if the post-infection time elapsed is less since there is lesstime for the bacteria to proliferate. In like manner an efficaciousdosage will depend on the bacterial load at the time of diagnosis.Multiple injections administered over a period of days could beconsidered for therapeutic usage.

The multivalent conjugates of the present invention can be administeredas a single dose or in a series (i.e., with a “booster” or “boosters”).For example, a child could receive a single dose early in life, then beadministered a booster dose up to ten years later, as is currentlyrecommended for other vaccines to prevent childhood diseases.

The booster dose will generate antibodies from primed B-cells, i.e., ananamnestic response. That is, the multivalent conjugate vaccine elicitsa high primary (i.e., following a single administration of vaccine)functional antibody response in younger populations when compared to thelicensed polysaccharide vaccine, and is capable of eliciting ananamnestic response (i.e., following a booster administration),demonstrating that the protective immune response elicited by themultivalent conjugate vaccine of the present invention is long-lived.

Compositions of the invention can include liquid preparations fororifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlinqual, alveolar, gingival, olfactory or respiratorymucosa) etc., administration such as suspensions, syrups or elixirs;and, preparations for parenteral, subcutaneous, intradermal,intramuscular, intraperitoneal or intravenous administration (e.g.,injectable administration), such as sterile suspensions or emulsions.Intravenous and parenteral administration are preferred. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose or thelike. The compositions can also be lyophilized. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Compositions of the invention are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions that may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or adose having a particular particle size.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally, to animals, children, particularly small children,and others who may have difficulty swallowing a pill, tablet, capsule orthe like, or in multi-dose situations. Viscous compositions, on theother hand, can be formulated within the appropriate viscosity range toprovide longer contact periods with mucosa, such as the lining of thestomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage for (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the active ingredient.Minor amounts of other ingredients such as pH adjusters (e.g., a basesuch as NaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, (e.g., methylcellulose),colors and/or flavors may also be present. The compositions can beisotonic, i.e., it can have the same osmotic pressure as blood andlacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium tartrate, propylene glycol or other inorganicor organic solutes. Sodium chloride is preferred particularly forbuffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount thatwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to theN. meningitidis polysaccharide-protein carrier conjugates.

The invention will be further described by reference to the followingillustrative, non-limiting examples setting forth in detail severalpreferred embodiments of the inventive concept. Other examples of thisinvention will be apparent to those skilled in the art without departingfrom the spirit of the invention.

EXAMPLES Example 1 Preparation of Neisseria meningitides Serogroups A,C, W-135, and Y Purified Capsular Polysaccharides Powders Crude PastePreparation

Separately, Neisseria meningitidis serogroup A, C, W135, and Y wetfrozen seed cultures were thawed and recovered with the aid of liquidWatson Scherp medium and planted in Blake bottles containing MuellerHinton agar medium. The Blake bottles were incubated at 35 to 37° C. ina CO₂ atmosphere for 15 to 19 hours. Following the incubation period,the growth from the Blake bottles were dislodged and added to 4L flaskscontaining Watson Scherp medium. The flasks were incubated at 35 to 37°C. for 3 to 7 hours on a platform shaker. The contents of the 4 L flaskswere transferred to a fermenter vessel containing Watson Scherp medium.The fermenter vessel was incubated at 35 to 37° C. for 7 to 12 hourscontrolling dissolved oxygen content and pH with supplement feed andantifoam additions. After the incubation period, the contents of thefermentor vessel were transferred to a 500 L tank, CETAVLON®(cetyltrimethyl-ammonium bromide; CTAB) was added, and the materialmixed for 1 hours. The Cetavlon treated growth was centrifuged atapproximately 15,000 to 17,000×g at a flow rate of approximately 30 to70 liters per hours. The crude polysaccharide was precipitated from thesupernatant with a second Cetavlon precipitation. Cetavlon was added tothe supernatant and the material mixed for at least 1 hour at roomtemperature. The material was stored at 1 to 5° C. for 8 to 12 hours.The precipitated polysaccharide was collected centrifugation atapproximately 45,000 to 50,000×g at a flow rate of 300 to 400 ml perminute. The collected paste was stored at −60° C. or lower until furtherprocessed.

Purified Polysaccharide Powder Preparation

The inactivated paste was thawed and transferred to a blender. The pastewas blended with 0.9M calcium chloride to yield a homogeneoussuspension. The suspension was centrifuged at approximately 10,000×g for15 minutes. The supernatant was decanted through a lint free pad into acontainer as the first extract. A second volume of 0.9M calcium chloridewas added to the paste, and blended to yield a homogeneous suspension.The suspension was centrifuged as above, and the supernatant combinedwith the supernatant from the first extraction. A total of fourextractions were performed, and the supernatants pooled. The pooledextracts were concentrated by ultrifiltration using 10-30 kDA MWCOspiral wound ultrafiltration units.

Magnesium chloride was added to the concentrated, and the pH adjusted to7.2 to 7.5 using sodium hydroxide. DNase and RNase were added to theconcentrate, and incubated at 25 to 28° C. with mixing for 4 hours.Ethanol was added to a concentration of 30 to 50%. Precipitated nucleicacid and protein were removed by centrifugation at 10,000×g for 2 hours.The supernatant was recovered and the polysaccharide precipitated byadding ethanol to 80% and allowing it to stand overnight at 1 to 5° C.The alcohol was siphoned off, and the precipitated polysaccharide wascentrifuged for 5 minutes at 10,000×g. The precipitated polysaccharidewas washed with alcohol. The polysaccharide was washed with acetone,centrifuged at 15 to 20 minutes at 10,000×g. The polysaccharide wasdried under vacuum. The initial polysaccharide powder was dissolved intosodium acetate solution. Magnesium chloride was added and the pHadjusted to 7.2 to 7.5 using sodium hydroxide solution. DNase and RNasewere added to the solution and incubated at 25 to 28° C. with mixing for4 hours to remove residual nucleic acids. After incubation with theseenzymes, an equal volume of sodium acetate-phenol solution was added tothe polysaccharide-enzyme mixture, and placed on a platform shaker at 1to 5° C. for approximately 30 minutes. The mixture was centrifuged at10,000×g for 15 to 20 minutes. The upper aqueous layer was recovered andsaved. An equal volume of sodium acetate-phenol solution was added tothe aqueous layer, and extracted as above. A total of four extractionswere performed to remove protein and endotoxin from the polysaccharidesolution. The combined aqueous extracts were diluted up to ten fold withwater for injection, and diafiltered against 10 volumes of water forinjection. Calcium chloride was added to the diafiltered polysaccharide.The polysaccharide was precipitated overnight at 1 to 5° C. by addingethanol to 80%. The alcohol supernatant was withdrawn, and thepolysaccharide collected by centrifugation at 10,000×g for 15 minutes.The purified polysaccharide was washed two times with ethanol, and oncewith acetone. The washed powder was dried under vacuum in a desiccator.The dried powder was stored at -30° C. or lower until processed ontoconjugate.

Example 2 Depolymerization of Neisseria meningitidis Serogroups A, C,W135, and Y Purified Capsular Polysaccharide Powder

Materials used in the preparation include purified capsularpolysaccharide powders from Neisseria meningitidis serogroups A, C,W-135, and Y (prepared in accordance with Example 1), sterile 50 mMsodium acetate buffer, pH 6.0, sterile 1N hydrocholoric acid, sterile 1Nsodium hydroxide, 30% hydrogen peroxide, and sterile physiologicalsaline (0.85% sodium chloride).

Each serogroup polysaccharide was depolymerized in a separate reaction.A stainless steel tank was charged with up to 60g of purified capsularpolysaccharide powder. Sterile 50 mM sodium acetate buffer, pH 6.0 wasadded to the polysaccharide to yield a concentration of 2.5 gpolysaccharide per liter. The polysaccharide solution was allowed to mixat 1 to 5° C. for 12 to 24 hours to effect solution. The reaction tankwas connected to a heat exchanger unit. Additional 50 mM sodium acetatebuffer, pH 6.0, was added to dilute the polysaccharide to reactionconcentration of 1.25 g per liter. The polysaccharide solution washeated to 55° C.±0.1. An aliquot of 30% hydrogen peroxide was added tothe reaction mixture to yield a reaction concentration of 1% hydrogenperoxide.

The course of the reaction was monitored by following the change in themolecular size of the polysaccharide over time. Every 15 to 20 minutes,aliquots were removed from the reaction mixture and injected onto aHPSEC column to measure the molecular size of the polysaccharide. Whenthe molecular size of the polysaccharide reached the targeted molecularsize, the heating unit was turned off and the polysaccharide solutionrapidly cooled to 5° C. by circulation through an ice water bath. Thedepolymerized polysaccharide solution was concentrated to 15 g perliters by connecting the reaction tank to an ultrafiltration unitequipped with 3000 MWCO regenerated cellulose cartridges. Theconcentrated depolymerized polysaccharide solution was diafilteredagainst 10 volumes of sterile physiological saline (0.85% sodiumchloride). The depolymerized polysaccharide was stored at 1 to 5° C.until the next process step.

The molecular size of the depolymerized polysaccharide was determined bypassage through a gel filtration chromatography column sold under thetradename “ULTAHYDROGEL™250” that was calibrated using dextran molecularsize standards and by multi-angle laser light scattering. The quantityof polysaccharide was determined by phosphorus content for serogroup Ausing the method of Bartlet, G. R. J. (1959) Journal of BiologicalChemistry, 234, pp-466-468, and by the sialic acid content forserogroups C, W135 and Y using the method of Svennerholm, L. (1955)Biochimica Biophysica Acta 24, pp604-611. The O-acetyl content wasdetermined by the method of Hesterin, S. (1949) Journal of BiologicalChemistry 180, p249. Reducing activity was determined by the method ofPark, J. T. and Johnson, M. J. (1949 Journal of Biological Chemistry181, pp149-151. The structural integrity of the depolymerizedpolysaccharide was determined by protein ¹H and ¹³C NMR. The purity ofthe depolymerized polysaccharide was determined by measuring the LAL(endotoxin) content and the residual hydrogen peroxide content.

Example 3 Derivatization of Neisseria meningitidis Serogroups A, C,W-135, and Y Depolymerized Polysaccharide

Materials used in this preparation include hydrogen peroxidedepolymerized capsular polysaccharide serogroups A, C, W-135, and Y fromNeisseria meningitidis (prepared in accordance with Example 2), adipicacid dihydrazide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC)for serogroup A only, sodium cyanborohydride, sterile 1N hydrocholoricacid, sterile 1N sodium hydroxide, sterile 1M sodium chloride, andsterile physiological saline (0.85% sodium chloride).

Each serogroup polysaccharide was derivatized in a separate reaction. Astainless steel tank was charged with the purified depolymerizedpolysaccharide, and diluted with sterile 0.85% physiological saline toachieve a final reaction concentration of 6 g polysaccharide per liter.To this solution was added a concentrated aliquot of adipic aciddihydrazide dissolved in sterile 0.85% physiological saline, in order toachieve a reaction concentration of 1 g per liter. For serogroup A only,EDAC was added as a concentrated aliquot dissolved in sterile 0.85%physiological saline, to achieve a reaction concentration of 1 g perliter. The pH was adjusted to 5.0±0.1, and this pH was maintained for 2hours using sterile 1N hydrochloric acid and sterile 1N sodium hydroxideat room temperature (15 to 30° C.). After two hours, a concentratedaliquot of sodium cyanoborohydride, dissolved in 0.85% physiologicalsaline, was added to the reaction mixture to achieve a reactionconcentration of 2 g per liter. The reaction was stirred at roomtemperature (15 to 30° C.) for 44 hours ±4 hours while maintaining thepH at 5.5±0.5. Following this reaction period, the pH was adjusted to6.0±0.1, and the derivatized polysaccharide was concentrated to 12 gpolysaccharide per liter by connecting the reaction tank to aultrafiltration unit equipped with a 3000 MWCO regenerated cellulosecartridges. The concentrated derivatized polysaccharide was diafilteredagainst 30 volumes of 1M sodium chloride, followed by 10 volumes of0.15M sodium chloride. The tank was disconnected from theultrafiltration unit and stored at 1 to 5° C. for 7 days. The tank wasreconnected to an ultrafiltration unit equipped with 3000 MWCOregenerated cellulose cartridges, and diafiltered against 30 volumes of1M sodium chloride, followed by 10 volumes of 0.15M sodium chloride.

The molecular size of the derivatized polysaccharide, the quantity ofpolysaccharide, and the O-acetyl content were measured by the samemethods used on the depolymerized polysaccharide. The hydrazide contentwas measured by the 2, 4, 6-trinitrobenzensulfonic acid method ofSnyder, S. L., and Sobocinski, P. Z. (1975) Analytical Biochemistry 64,pp282-288. The structural integrity of the derivatized polysaccharidewas determined by proton ¹H and ¹³C NMR. The purity of the derivatizedpolysaccharide was determined by measuring the level of unboundhydrazide, the LAL (endotoxin) content, and the residualcyanoborohydride content.

Example 4 Preparation of Carrier Protein Preparation of Crude DiphtheriaToxoid Protein

Lyophilized seed cultures were reconstituted and incubated for 16 to 18hours. An aliquot from the culture was transferred to a 0.5-liter flaskcontaining growth medium, and the culture flask was incubated at 34.5 to36.5° C. on a rotary shaker for 7 to 9 hours. An aliquot from theculture flask was transferred to a 4-liter flask containing growthmedium, and the culture flask was incubated at 34.5 to 36.5° C. on arotary shaker for 14 to 22 hours. The cultures from the 4-liter flaskwere used to inoculate a fermenter containing growth media. Thefermenter was incubated at 34.5 to 36.5° C. for 70 to 144 hours. Thecontents of the fermenter were filtered through depth filters into acollection vessel. An aliquot of formaldehyde solution, 37% was added tothe harvest to achieve a concentration of 0.2%. The pH was adjusted to7.4 to 7.6. The harvest was filtered through a 0.2 micron filtercartridge into sterile 20 liter bottles. The bottles were incubated at34.5 to 36.5° C. for 7 days. An aliquot of formaldehyde solution, 37%,was added to each 20 liter bottle to achieve a concentration of 0.4%.The pH of the mixtures was adjusted to 7.4 to 7.6. The bottles wereincubated at 34.5 to 36.5° C. for 7 days on a shaker. An aliquot offormaldehyde solution, 37%, was added to each 20 liter bottle to achievea concentration of 0.5%. The pH of the mixtures was adjusted to 7.4 to7.6. The bottles were incubated at 34.5 to 36.5° C. for 8 weeks. Thecrude toxoid was tested for detoxification. The bottles were stored at 1to 5° C. during the testing period.

Purification of the Crude Diphtheria Toxoid Protein

The crude toxoid was allowed to warm to room temperature, and thecontents of the 20-liter bottles were combined into a purification tank.The pH of the toxoid was adjusted to 7.2 to 7.4, and charcoal was addedto the crude toxoid and mixed for 2 minutes. The charcoal toxoid mixturewas allowed to stand for 1 hours, and was then filtered through a depthfilter cartridge into a second purification tank. Solid ammonium sulfatewas added to the filtrate to achieve 70% of saturation. The pH wasadjusted to 6.8 to 7.2, and the solution was allowed to stand for 16hours. The precipitated protein was collected by filtration and washedwith 70% of saturation ammonium sulfate solution, pH 7.0. Theprecipitate was dissolved into sterile distilled water, and the proteinsolution was filtered into a stainless steel collection vessel. The pHwas adjusted to 6.8 to 7.2, and ammonium sulfate was added to 40% ofsaturation. The pH of the solution was adjusted to 7.0 to 7.2, and thesolution was allowed to stand for 16 hours. The precipitate was removedby filtration and discarded. Ammonium sulfate was added to the filtrateto 60% of saturation, and the pH adjusted to 7.0 to 7.2. The mixture wasallowed to stand for 16 hours, and the precipitated protein wascollected by filtration. The precipitate was dissolved into steriledistilled water, filtered to remove undissolved protein, and diafilteredagainst 0.85% physiological saline.

Concentration and Sterile Filtration of the Purified Diphtheria ToxoidProtein

The protein solution was concentrated to 15g per liter and diafilteredagainst 10 volumes of 0.85% physiological saline suing a 10,000 MWCOregenerated cellulose filter cartridge. The concentrated proteinsolution was sterilized by filtration through a 0.2 micron membrane. Theprotein solution was stored at 1 to 5° C. until processed ontoconjugate.

The protein concentration was determined by the method of Lowry, O. H.et. al (1951) Journal of Biological Chemistry 193, p265-275. The purityof the protein was measured by sterility, LAL (endotoxin) content, andresidual formaldehyde content.

Example 5 Preparation of Monovalent Conjugates of Neisseria meningitidisSerogroups A, C, W-135, and Y Polysaccharide to Diphtheria ToxoidProtein

Materials used in this preparation include adipic acid derivatizedpolysaccharide from Neisseria meningitidis serogroups A, C, W-135, and Y(prepared in accordance with Example 3), sterile diphtheria toxoidprotein (prepared in accordance with Example 4), EDAC, ammonium sulfate,sterile 1N hydrochloric acid, sterile 1N sodium hydroxide, and sterilephysiological saline (0.85%).

Each serogroup polysaccharide conjugate was prepared by a separatereaction. All four conjugates were prepared by the following process. Astainless steel tank was charged with the purified adipic acidderivatized polysaccharide at a reaction concentration of 700 to 1000μmoles of reactive hydrazide per liter and purified diphtheria toxoidprotein at a reaction concentration of 3.8 to 4.0 g protein per liter.Physiological saline 0.85%, was used to dilute the starting materials tothe target reaction concentrations and the pH was adjusted to 5.0±0.1.An aliquot of EDAC was added to the polysaccharide protein mixture toachieve a reaction concentration of 2.28 to 2.4 g per liter. The pH ofthe reaction was kept at 5.0±0.1 for 2 hours at 15 to 30° C. After twohours, the pH was adjusted to 7.0±0.1 using sterile 1N sodium hydroxide,and the reaction was stored at 1 to 5° C. for 16 to 20 hours.

The reaction mixture was allowed to warm to 15 to 30° C. and thereaction vessel was connected to an ultrafiltration unit equipped with a30,000 MWCO regenerated cellulose cartridge. Solid ammonium sulfate wasadded to 60% of saturation (for serogroups A, W-135 and Y) and 50% ofsaturation (for serogroup C). The conjugate reaction mixture wasdiafiltered against 20 volumes of 60% of saturated ammonium sulfatesolution (for serogroups A, W-135 and Y) and 50% of saturated ammoniumsulfate solution (for serogroup C), followed by 20 volumes ofphysiological saline, 0.85%. The diafiltered conjugate was firstfiltered through a filter capsule containing a 1.2 micron and a 0.45micron filter, and then through a second filter capsule containing a0.22 micron filter.

The quantity of polysaccharide and O-acetyl content were measured by thesame methods used on the depolymerized and derivatized polysaccharide.The quantity of protein was determined by the Lowry method. Themolecular size of the conjugate was determined by passage through a gelfiltration chromatography column sold under the tradename “TSK6000PW”that used DNA as the void volume marker, ATP as the total volume marker,and bovine thyroglobulin as a reference marker. In addition, themolecular size of the conjugate eluted from the TKS6000PW column wasmeasured by multi-angle laser light scattering. The antigenic characterof the conjugate was measured by binding to anti-polysaccharideserogroup specific antibody using double-sandwich ELISA method. Thepurity of the conjugates was determined by measuring the amount ofunbound (unconjugated) polysaccharide by elution though a hydrophobicinteraction chromatography column, unconjugated protein by capillaryelectrophoresis, sterility, LAL (endotoxin) content, residual EDACcontent, and residual ammonium ion content.

Example 6 Formulation of a Multivalent Meningococcal A, C, W-135, and YPolysaccharide Diphtheria Toxoid Conjugate Vaccine

Materials used in this preparation include, serogroups A, C, W-135, andY polysaccharide-diphtheria toxoid conjugates (prepared in accordancewith Example 5), sterile 100 mM sodium phosphate buffered physiologicalsaline (0.85% sodium chloride).

An aliquot of sterile 100-500 mM sodium phosphate buffered physiologicalsaline was added to physiological saline (0.85%) in a stainless steelbulking tank to yield a final vaccine concentration of 10 mM sodiumphosphate. An aliquot of each of from two to four of the sterilemonovalent meningococcal polysaccharide-diphtheria toxoid conjugates wasadded to the bulking tank containing 10 mM sterile sodium phosphatephysiological saline to yield a final concentration of 8 μg of eachserogroup polysaccharide per milliliter of buffer. The formulatedtetravalent conjugate was mixed and filtered through a 0.2 μm filterinto a second bulking tank.

The quantity of each serogroup polysaccharide present in the multivalentformulation was determined by component saccharide analysis using highpH anion-exchange chromatography with pulsed amperometric detection. Thequantity of protein was measured by the method of Lowry. The pH of thevaccine was measured using a combination electrode connected to a pHmeter. The antigenic character of the multivalent conjugate vaccine wasmeasured by binding to anti-polysaccharide serogroup specific antibodyusing a double-sandwich ELISA method. Immunogenicity of the multivalentconjugate vaccine was measured the ability of each conjugate present inthe vaccine to elicit both a primary and booster anti-polysaccharide IgGimmune response in an animal model. The purity of the multivalentconjugate vaccine was determined by measuring the amount of unbound(unconjugated) polysaccharide using high pH anion-exchangechromatography with pulsed amperometric detection, sterility, LAL(endotoxin) content, pyrogenic content, and general safety.

Example 7 Preparation of Aluminum-Hydroxide Adjuvanted MultivalentMeningococcal Polysaccharide Diphtheria Toxoid Protein Conjugate

Preparation of conjugate adsorbed to aluminum hydroxide. Materials usedin this preparation include serogroups A, C, W-135, and Ypolysaccharide-diphtheria toxoid conjugates preparation described inExample 5, sterile physiological saline (0.85% sodium chloride), andsterile aluminum hydroxide in physiological saline (0.85% sodiumchloride).

An aliquot of each of the sterile monovalent meningococcalpolysaccharide diphtheria toxoid conjugates was added to the bulkingtank containing physiological saline to yield a final concentration of 8μg of each serogroup polysaccharide per milliliter of buffer. An aliquotof sterile aluminum hydroxide in physiological saline (0.85% sodiumchloride) was added to the multivalent conjugate vaccine to achieve afinal concentration of 0.44 mg aluminum ion per milliliter vaccine.

Example 8 Preparation of Aluminum Phosphate-Adjuvanted Conjugate

Materials used in this preparation include serogroups A, C, W-135, and Ypolysaccharide-diphtheria toxoid conjugates preparation described inExample 5, sterile physiological saline (0.85% sodium chloride), andsterile aluminum phosphate in physiological saline (0.85% sodiumchloride).

An aliquot of each of the sterile monovalent meningococcalpolysaccharide-diphtheria toxoid conjugates was added to the bulkingtank containing physiological saline to yield a final concentration of 8μg of each serogroup polysaccharide per milliliter of buffer. An aliquotof sterile aluminum phosphate in physiological saline (0.85% sodiumchloride) was added to the multivalent conjugate vaccine to achieve afinal concentration of 0.44 mg aluminum on per milliliter vaccine.

Example 9 Immunogenicity of the Tetravalent Conjugate Vaccine

The tetravalent conjugate vaccine was studied for its ability to elicitan immune response in small laboratory animals prior to evaluation inthe clinic. Mice, rats and rabbits have been used to study theimmunogenicity of conjugate vaccines relative to the polysaccharidevaccines. These animal models are useful, because they are capable ofdistinguishing the conjugate vaccine from the correspondingpolysaccharide by their immune response pattern. The conjugate vaccineelicits a T-dependent-like immune response in these models, whereas thepolysaccharide vaccine elicits a T-independent-like immune response.

In the mouse immunogenicity studies, the conjugate was diluted withphysiological saline (0.85% sodium chloride) to administer betweenone-quarter to one-sixteenth of a human dose. The mice were administeredone or two doses of vaccine, either conjugate or polysaccharide, andblood specimens were taken two weeks post vaccination. One group of miceserved as an unimmunized control group. Antibodies to each of theserogroup polysaccharides were measured by an ELISA method. The serumsamples were incubated with excess of each capsular polysaccharide thatwas bound to a ELISA microtiter plate well. Methylated human serumalbumin was used to bind each serogroup polysaccharide to the microtiterwell. Following incubation the microtiter well was washed with buffer,and a secondary antibody-enzyme conjugate was added to theantibody-polysaccharide complex which binds to the anti-meningococcalpolysaccharide antibody. The microtiter plate was washed, and a chemicalsubstrate was added to the polysaccharide-meningococcalantibody-secondary antibody-enzyme conjugate. The enzyme hydrolyzes aportion of the chemical substrate that results in color formation. Theamount of color formation is proportional to the amount ofpolysaccharide-meningococcal antibody-secondary antibody-enzymeconjugate that is bound to the microtiter well. The potency of thevaccine was determined by comparison to reference antisera for eachserogroup, which is measured in the same microtiter plate, by a parallelline calculation using a four-parameter fit. The mouse referenceantisera was generated in the same strain of mice that were individuallyimmunized with three doses of each serogroup conjugate vaccine. Themouse reference antisera were assigned titers based on the inverse ofdilution yielding an optical density of 1.0.

Presented in Table 1 is a summary of anti-polysaccharide IgG titers foreach serogroup achieved in Swiss-Webster mice who were vaccinated withtwo doses of either the tetravalent conjugate vaccine, both liquid andaluminum hydroxide formulation, or the corresponding tetravalentpolysaccharide vaccine. The IgG titers were measured on pooled sera froma set of ten mice. Two sets of 10 mice were used to measure the immuneresponse to each vaccine formulation. Both sets were vaccinated onday 1. On day 15 (2 weeks post vaccination) blood specimens were takenfrom one set of 10 mice, and the second set of ten mice were vaccinatedwith a second dose of vaccine on day 15. Two weeks late on day 29, bloodspecimens were taken from the second set of 10 mice, and from theunimmunized control group. All antibodies were titrated at the sametime, that is, both the day 15 and day 29 blood specimens were assayedat the same time along with the unimmunized controls and the mousereference sera.

TABLE 1 Anti-polysaccharide IgG titers on pooled sera from Swiss-Webstermice vaccinated with either tetravalent conjugate or polysaccharide.Anti-Men Anti Men Vaccine Dosage A Anti-Men C W135 Anti-Men Y Group μgps D15 D29 D15 D29 D15 D29 D15 D29 Conjugate 0.25 131 2640 250 1510 13506100 5660 4830 (no adjuvant) Conjugate 0.50 171 6220 416 2050 849 260005980 112000 (no adjuvant) Conjugate 1.0 249 4500 525 2740 1450 1660011300 59100 (no adjuvant) Conjugate 0.25 2920 4500 1010 2980 2300 3370011600 124000 (Alum. Hyd.) Conjugate 0.50 5800 9550 2280 1010 4810 7190026400 330000 (Alum Hyd.) Conjugate 1.0 6210 9350 2630 12800 7870 9400032700 302000 (Alum Hyd.) Polysaccharide 1.0 136 173 184 205 612 608 44703910 (no adjuvant) Unimunized n.a. — 110 — 145 — 623 — 777

The tetravalent conjugate vaccine, both unadjuvanted and adjuvanted withaluminum hydroxide, is capable of eliciting a strong anti-polysaccharideIgG immune response in this mouse model. The aluminum hydroxide adjuvantserves to improve both the primary and booster response to each of thefour serogroup polysaccharide conjugates. The tetravalent polysaccharidevaccine elicits a negligible immune response to serogroups A, C, andW135 in this mouse model relative to the unimmunized control, whereasserogroup Y does elicit a respectable immune response, but not a boosterresponse. The tetravalent polysaccharide vaccine fails to elicit abooster response to all four serogroup polysaccharides in this model.This model can readily differentiate between the polysaccharide vaccineand the conjugate vaccine both by the magnitude of the immune responseand booster response pattern to each of the serogroup conjugatevaccines.

The unadjuvanted form of the tetravalent conjugate vaccine has beenstudied in the clinic for safety and immunogenicity in young healthyadults and in young healthy children. In the adult study, subjects werevaccinated with a single dose of vaccine, formulated to contain 4 μg ofeach of the four conjugates, as polysaccharide. Blood specimens weretaken immediately prior to vaccination and 28-days post vaccination.Antibodies to each of the serogroup conjugates were measured by an ELISAmeasurement that quantified the amount of anti-polysaccharide IgG. TheELISA method is very similar to the method used to measure the amount ofIgG antibody present in mouse sera.

Briefly, the serum samples were incubated in ELISA microtiter wells thatwere coated with excess meningococcal polysaccharide that was bound tothe plate with methylated human serum albumin. The amount of boundantibody was determined by a reaction with peroxidase-labeled mouseanti-human IgG specific monoclonal antibody. A subsequent reaction usingperoxidase substrate generates a chromogenic product that was measuredspectrophotometrically. The resulting optical density of the chromophorecorrelates with the amount of IgG antibody in the serum that is bound tothe meningococcal polysaccharide on the microtiter plate. The amount ofantibody was calculated by comparison to a human reference sera (CDC1922) with an assigned value using a 4-parameter logistic curve method.In addition, the antibodies were measured for their ability to lyseserogroup specific bacteria. The serum samples are firstheat-inactivated to destroy complement. The serum samples are diluted bytwo-fold dilutions in a sterile 96-well microtiter plate. Serogroupspecific bacteria along with baby rabbit complement were added to theserum dilutions and allowed to incubate. After an incubation period, anagar overlay medium was added to the serum/complement/bacteria mixture.The agar overlay was allowed to harden, and then incubated overnight at37° C. with 5% carbon dioxide. The next day, bacterial colonies presentin the wells were counted. The endpoint titer was determined by thereciprocal serum dilution yielding greater than 50% killing as comparedto the mean of the complement control wells.

Presented in Table 2 is a summary of the anti-polysaccharide mean IgGconcentrations for each serogroup and the mean serum bactericidalantibody (SBA) titer in adult sera pre and post vaccination with thetetravalent conjugate vaccine formulated at 4 μg polysaccharide perdose. The immune response to all four serogroup conjugates weresatisfactory, that is comparable to the immune response achieved by thelicensed polysaccharide vaccine in terms of both IgG antibody andfunctional bactericidal antibody responses. The vaccine was found to besafe for this age group and the safety profile was found to be similarto that of the licensed polysaccharide vaccine.

TABLE 2 Anti-polysaccharide IgG GMC (group mean concentration and SerumBactericidal Antibody GMTs (group mean titers) for young healthy adultsvaccinated with a tetravalent meningococcal conjugate vaccine formulatedat 4 μg per dose by polysaccharide. Immune IgG GMC (μg/ml) Response byN_(pre)/ [95% CI] SBA GMT [95% CI] Serogroup N_(post) Pre Post Pre PostA 28/28 3.3 38.4 487 6720 [2.3-4.8] [22.2-66.4]  [231-1027] [4666-15428]C 28/28 0.4 5.5 16.4 1560 [0.2-0.7] [3.0-10.1] [7.1-37.7] [800-4042]W-135 28/28 0.6 5.8 10.0 609 [0.3-1.0] [2.9-11.7] [5.9-16.9] [250-1481]Y 28/28 1.3 6.8 19.0 390 [0.7-2.5] [3.2-14.6] [8.0-41.2] [143-1061]

In younger age groups, children less than 2 years of age, the immuneresponse to the polysaccharide vaccine is weak and the immunity has beenestimated to wane after one year. Children 12 to 15 months of age wereadministered a single dose of tetravalent conjugate vaccine formulatedat 4 μg of each serogroup polysaccharide per dose, and they wereadministered a second dose of tetravalent conjugate vaccine two monthsfollowing the first dose. Blood specimens were taken prior to the firstand second vaccination, and one month post the second vaccination. Theantibody responses to the four serogroup conjugates are summarized inTable 3. For each serogroup a booster response for IgG antibody and forfunctional-bactericidal antibody was observed following a second dose oftetravalent conjugate. The level of IgG antibody elicited by theconjugate vaccine is comparable to that elicited by the licensedpolysaccharide for this age group; a post 6 week response of 3.64 μg/ml(2.96-4.49) IgG antibody response to serogroup C polysaccharide.However, the level of bactericidal antibody elicited by the conjugatevaccine is much higher than what is normally elicited by the licensedpolysaccharide vaccine for this age group; a post 6 week SBA titer of7.2 (5.0-10.4). The reason for this discordance between IgG antibody andbactericidal antibody in the younger populations is thought to resultfrom the polysaccharide eliciting a high proportion of low avidityantibody in the younger populations. Conversely, the conjugate appearsto elicit a much higher proportion of high avidity antibody. Highavidity antibody is thought to be responsible for the bactericidalactivity.

TABLE 3 Anti-polysaccharide IgG GMC (group mean concentration) and SerumBactericidal Antibody GMTs (group mean titers) for young healthychildren (1 to 2 years of age) vaccinated with two doses of tetravalentmeningococcal conjugate vaccine formulated at 4 μg per dose bypolysaccharide Immune IgG GMC Response (μg/ml) [95% CI] SBA GMT [95% CI]by Pre Pre Post Pre Pre Serogroup N₁/N₂/N₃ dose 1 Dose 2 Dose 2 Dose 1Dose 2 Post dose 2 A 8/8/8 0.2 2.1 4.4 8.7 1328    3158  [0.1-0.4][0.9-4.8] [2.1-9.1] [1.4-55.1] [179-9871] [1857-5371] C 8/8/8 0.2 1.01.5 6.7 117   304 [0.0-0.7] [0.3-3.1] [0.6-3.6] [2.0-23.0] [37.7-365][128-721] W-135 8/8/8 0.1 0.6 1.5 6.2 22.6 430 [0.1-0.2] [0.2-1.9][0.8-3.1] [2.2-17.2] [2.8-185] [172-1076] Y 8/8/8 0.3 1.2 4.5 5.7 98.7304 [0.2-0.4] [0.5-2.8] [2.7-7.6] [3.7-8.8] [20.4-478] [101-920]

In addition to the ability of the tetravalent conjugate vaccine toelicit a high functional antibody response in younger populationscompared to the licensed polysaccharide vaccine, the tetravalentconjugate vaccine is capable of eliciting an anamnestic response,demonstrating that protection elicited by the tetravalent conjugatevaccine of the present invention is long-lived. In the development ofthe tetravalent conjugate vaccine, studies were first conducted on abivalent AC conjugate formulation. The vaccine offers wider coveragethan the current licensed monovalent C conjugate, but does not protectagainst disease caused by serogroups W135 and Y.

A clinical study was performed with infant subjects that compared theimmune response to the bivalent AC polysaccharide vaccine versus thebivalent AC conjugate vaccine. In this study, a third group of infantswere enrolled to serve as a control group and they received aHaemophilus influenzae type b conjugate. All three vaccine groupsreceive the same pediatric vaccines. The bivalent AC conjugate groupreceived three doses of conjugate vaccine (4 μg polysaccharide per dose)at 6, 10, and 14 weeks of age. The bivalent AC polysaccharide groupreceived two doses of a bivalent AC polysaccharide vaccine (50 μgpolysaccharide per dose) at 10 and 14 weeks of age. The Haemophilusinfluenzae type b conjugate group received three doses of conjugatevaccine at 6, 10, and 14 weeks of age. Blood specimens were taken at 6weeks, pre-vaccination, and at 18 weeks, 4 weeks post vaccination. Whenthe children were 11 to 12 months of age, blood specimens were taken andthe children who had received either the bivalent AC conjugate or thebivalent AC polysaccharide vaccine received a booster dose of ACpolysaccharide. The reason for the booster dose of polysaccharide was toevaluate whether or not the subjects would elicit an anamnesticresponse.

The results of this study, both the primary and polysaccharide boosterimmune responses are presented in Table 4 for the IgG antibody responseand Table 5 for the SBA antibody response. The IgG antibody responsepost primary series was approximately the same for both thepolysaccharide and conjugate vaccine. However, the bactericidal antibodyresponse in the conjugate vaccinated subjects was much higher than thatfor the polysaccharide vaccinated subjects. As observed with the oneyear old subjects, vaccination of infants with the polysaccharideelicits very little functional-bactericidal antibody. The antibodyelicited by the infants to the polysaccharide vaccine is presumably lowavidity antibody, whereas, the conjugate vaccine appears to elicit highavidity antibody, thereby accounting for the much higher titer ofbactericidal antibody. The high level of functional antibody elicited bythe booster dose of polysaccharide vaccine in the subjects who hadreceived the conjugate vaccine in the primary vaccination series,indicates that these subjects have been primed for a memory or T-celldependent antibody response. The subjects who received thepolysaccharide vaccine in the primary vaccination series elicited amodest response to the polysaccharide booster dose, that is indicativeof a T-cell independent response.

TABLE 4 Anti-polysaccharide IgG GMC (group mean concentration) ininfants against serogroups A and C before and after both the primaryseries immunization (6, 10 and 14 weeks of age) and the boostervaccination with bivalent AC polysaccharide given at 11 to 12 months ofage. Immune Response by Primary Vaccination GMC PS Booster VaccinationVaccine [95% CI] GMC [95% CI] Group N Pre Post N Pre Post Serogroup A:AC 34 3.4 5.8 31 0.2 7.0 Conjugate [2.2-5.4] [4.3-8.0] [0.1-0.3][4.0-12.0] AC 35 3.0 5.5 30 0.9 3.1 Poly- [1.7-5.3] [4.1-7.3] [0.5-1.4][2.0-4.7] saccharide HIB 36 3.2 0.6 NA NA NA Conjugate [2.2-4.5][0.4-0.8] Serogroup C: AC 31 1.6 2.8 31 0.1 8.1 Conjugate [0.9-2.8][2.0-3.9] [0.1-0.2] [4.5-14.5] AC 35 2.3 5.3 30 0.6 2.8 Poly- [1.4-3.9][3.8-7.4] [0.3-1.0] [1.7-4.7] saccharide HIB 36 2.0 0.5 NA NA NAConjugate [1.2-3.5] [0.3-0.7]

TABLE 5 SBA antibody GMT (group mean titer) in infants againstserogroups A and C before and after both the primary series immunization(6, 10 and 14 weeks of age) and booster vaccination with bivalent ACpolysaccharide given at 11 to 12 months of age. Primary Vaccination GMTPS Booster Vaccination Immune Response by [95% CI] GMT [95% CI] VaccineGroup N Pre Post N Pre Post Serogroup A: AC Conjugate 34 11.8 177    2410.1  373 [7.2-19.3] [101-312]  [5.6-18.0] [162-853] AC Polysaccharide32 14.7 7.0 26 6.1   24.1 [8.5-25.4]  [4.7-10.5] [3.9-9.5] [11-53] HIBConjugate 35 11.2 6.7 NA NA NA [6.8-18.3]  [4.3-10.5] Serogroup C: ACConjugate 34 50.8 189    27 4.6 287 [24-107] [128-278] [3.6-5.6][96.2-858]  AC Polysaccharide 32 62.7 25.4  26 4.1   14.4 [29-131][14.4-44.6] [3.9-4.3]  [7.9-26.1] HIB Conjugate 36 45.3 7.3 NA NA NA[21.9-133]    [4.7-11.3]

In addition to the benefits that this invention offers to the improvedprotection against meningococcal disease in young populations and thewider protection against serogroups A, C, W-135 and Y, the tetravalentconjugate may provide protection to other pathogens by inducing anantibody response to the carrier protein. When the tetravalent conjugatevaccine, using diphtheria toxoid conjugate, was administered to infants,these subjects also received the routine pediatric immunizations, whichincluded diphtheria toxoid. Therefore, in these subjects there was noapparent improvement in the antibody response to diphtheria toxoid.However, when the diphtheria toxoid conjugate was administered tosubjects that did not receive concomitant diphtheria toxoid containingvaccines, a strong booster response to diphtheria toxoid was observed.These subjects had received a three dose regimen of DTP at 2, 3, and 4months of age. In this study, the subjects received either single doseof a bivalent AC conjugate or a single dose of bivalent ACpolysaccharide vaccine between 2 and 3 year of age. Blood specimens weretaken at the time of vaccination and 30-days post vaccination. Thebivalent AC conjugate used diphtheria toxoid as the carrier protein.

The immune response of diphtheria toxoid in the two vaccine groups ispresented in Table 6. The polysaccharide did not serve to stimulate ananti-diphtheria immune response in these subjects as expected, however astrong anti-diphtheria immune response was observed for the subjectsreceiving the AC conjugate. Therefore, the meningococcal conjugatevaccine may provide an added benefit of stimulating an immune responseto carrier protein thereby providing protection against diseases causedby Corynebacteria diphtherias when diphtheria toxoid is used as acarrier protein.

TABLE 6 Anti-diphtheria antibody by ELISA GMT (group mean titer) inIU/ml in young healthy children vaccinated with either a bivalent ACdiphtheria toxoid conjugate vaccine formulated at 4 μg as polysaccharideper dose or a bivalent AC polysaccharide vaccine formulated at 50 μg aspolysaccharide per dose Anti-Diptheria Antibody Immune Response by(ELISA-IU/ml) [95% CI] Vaccine Group N_(pre)/N_(post) Pre Post ACConjugate 104/103 0.047 21.2 [0.036-0.060] [11.6-38.6] AC Polysaccharide103/102 0.059 0.059 [0.045-0.076] [0.045-0.077]

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1.-17. (canceled)
 18. A method of manufacturing an immunologicalcomposition capable of eliciting in children less than two years of age,a primary serum IgG response and a serum bactericidal antibody responseto each of Neisseria meningitidis serogroups A, C, W-135 and Y capsularpolysaccharides, the method comprising the steps of: (a) purifyingcapsular polysaccharides from each of the N. meningitidis serogroups A,C, W-135 and Y; (b) partially depolymerizing the purified capsularpolysaccharides from each of the N. meningitidis serogroups A, C, W-135and Y in a separate reaction; (c) conjugating each of the purifiedcapsular polysaccharides of step (b) to a purified single carrierprotein species in a separate reaction, wherein four distinct andseparately made carrier protein-capsular polysaccharide conjugates aremade: (d) mixing the four distinct and separately made carrierprotein-capsular polysaccharide conjugates of step (c); (e) purifyingthe admixture of the four distinct and separately made carrierprotein-capsular polysaccharide conjugates, wherein the carrier proteinis selected from the group consisting of diphtheria toxoid, CRM₁₉₇ andtetanus toxoid, and further wherein, the serum bactericidal antibodyresponse in children less than two years of age is higher than thatelicited by the licensed, unconjugated, tetravalent A, C, W-135 and Ymeningococcal capsular polysaccharide vaccine.
 19. The method of claim18, wherein the immunological composition further comprises an adjuvant.20. The method of claim 19, wherein the adjuvant is an aluminumadjuvant.
 21. The method of claim 20, wherein the aluminum adjuvant isaluminum hydroxide.
 22. The method of claim 20, wherein the aluminumadjuvant is aluminum phosphate.
 23. The method of claim 18, wherein thecarrier protein is diphtheria toxoid.
 24. The method of claim 18,wherein the carrier protein is CRM₁₉₇.
 25. The method of claim 18,wherein the carrier protein is tetanus toxoid.
 26. A method ofmanufacturing a conjugate vaccine capable of eliciting in children lessthan two years of age, a primary serum IgG response and a serumbactericidal antibody response to each of Neisseria meningitidisserogroups A, C, W-135 and Y capsular polysaccharides, the methodcomprising the steps of: (a) purifying capsular polysaccharides fromeach of the N. meningitidis serogroups A, C, W-135 and Y; (b) partiallydepolymerizing the purified capsular polysaccharides from each of the N.meningitidis serogroups A, C, W-135 and Y in a separate reaction; (c)conjugating each of the purified capsular polysaccharides of step (b) toa purified single carrier protein species in a separate reaction,wherein four distinct and separately made carrier protein-capsularpolysaccharide conjugates are made: (d) mixing the four distinct andseparately made carrier protein-capsular polysaccharide conjugates ofstep (c); (e) purifying the admixture of the four distinct andseparately made carrier protein-capsular polysaccharide conjugates,wherein the carrier protein is selected from the group consisting ofdiphtheria toxoid, CRM₁₉₇ and tetanus toxoid, and further wherein, theserum bactericidal antibody response in children less than two years ofage is higher than that elicited by the licensed, unconjugated,tetravalent A, C, W-135 and Y meningococcal capsular polysaccharidevaccine.
 27. The method of claim 26, wherein the conjugate vaccinefurther comprises an adjuvant.
 28. The method of claim 27, wherein theadjuvant is an aluminum adjuvant.
 29. The method of claim 28, whereinthe aluminum adjuvant is aluminum hydroxide.
 30. The method of claim 28,wherein the aluminum adjuvant is aluminum phosphate.
 31. The method ofclaim 26, wherein the carrier protein is diphtheria toxoid.
 32. Themethod of claim 26, wherein the carrier protein is CRM₁₉₇.
 33. Themethod of claim 26, wherein the carrier protein is tetanus toxoid.
 34. Amethod of vaccinating a human susceptible to infection due to Neisseriameningitidis comprising administering to the human an immunologicallyeffective amount of an immunological composition comprising a mixture offour distinct and separately made protein-capsular polysaccharideconjugates, wherein the first conjugate comprises purified N.meningitidis capsular polysaccharide of serogroup W-135 conjugated to apurified carrier protein, the second conjugate comprises purified N.meningitidis capsular polysaccharide of serogroup Y conjugated to apurified carrier protein, the third conjugate comprises purified N.meningitidis capsular polysaccharide of serogroup A conjugated to apurified carrier protein, and the fourth conjugate comprises purified N.meningitidis capsular polysaccharide of serogroup C conjugated to apurified carrier protein, wherein the immunological composition has thecapacity to elicit in children less than two years of age a primaryserum IgG response and a serum bactericidal antibody response to each ofthe N. meningitidis serogroups A, C, W-135 and Y capsularpolysaccharides, wherein said serum bactericidal antibody response ishigher than that elicited by the licensed, unconjugated, tetravalent A,C, W-135 and Y meningococcal capsular polysaccharide vaccine.
 35. Themethod of claim 34, wherein the immunological composition furthercomprises an adjuvant.
 36. The method of claim 35, wherein the adjuvantis an aluminum adjuvant.
 37. The method of claim 36, wherein theadjuvant is aluminum hydroxide.
 38. The method of claim 36, wherein theadjuvant is aluminum phosphate.
 39. The method of claim 34, wherein saidcarrier protein is a single carrier protein species.
 40. The method ofclaim 34, wherein said single carrier protein species is selected fromthe group consisting of diphtheria toxoid, CRM₁₉₇ and tetanus toxoid.41. The method of claim 40, wherein said carrier protein species isdiphtheria toxoid.
 42. The method of claim 40, wherein said carrierprotein species is CRM₁₉₇.
 43. The method of claim 40, wherein saidcarrier protein species is tetanus toxoid.
 44. The method of claim 34,wherein the immunological composition is formulated as a sterile liquid.45. The method of claim 34, wherein the immunological compositionfurther comprises a pharmaceutically acceptable preservative.
 46. Themethod of claim 45, wherein said pharmaceutically acceptablepreservative is thimerosal.
 47. The method of claim 34, wherein themethod includes the administration of a primary dose and a booster doseof the immunological composition.
 48. The method of claim 34, whereinthe immunological composition is formulated at 4 microgram of each ofthe N. meningitidis serogroups A, C, W-135 and Y capsularpolysaccharides per dose of the composition.
 49. The method of claim 34,wherein the immunological composition is unadjuvanted.